1
The Bromodomains of the mammalian SWI/SNF (mSWI/SNF) ATPases Brahma (BRM) and 1
Brahma Related Gene 1 (BRG1) promote chromatin interaction and are critical for skeletal 2
muscle differentiation 3
4
Tapan Sharmaa, Hanna Witwickaa*, Anthony N. Imbalzanoa,# 5
6
a Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical 7
School, Worcester, MA 01605 USA 8
9
Running Title: BRG1/BRM bromodomains are critical for myogenesis 10
11
# Address correspondence to Anthony N. Imbalzano, [email protected]. 12
*Present Address: Hanna Witwicka, Charles River Laboratories, Inc., Shrewsbury, MA 01545, 13
USA 14
15
Word count for Abstract: 200 16
Word count for Introduction, Results and Discussion: 4901 (combined upper limit is 8000 words) 17
Word count for Materials and Methods: 1565 (no limit) 18
19
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ABSTRACT: Skeletal muscle differentiation induces changes in the epigenome of myoblasts as 20
they proceed towards a myogenic phenotype. mSWI/SNF chromatin remodeling enzymes 21
coordinate with lineage-determining transcription factors and are key regulators of differentiation. 22
Three mSWI/SNF proteins, the mutually exclusive ATPases, BRG1 and BRM, and the BAF180 23
protein (Polybromo1, PBRM1) contain bromodomains belonging to the same structural subfamily. 24
Bromodomains bind to acetylated lysines on histone N-terminal tails and on other proteins. 25
Pharmacological inhibition of mSWI/SNF bromodomain function using the selective inhibitor 26
PFI-3 reduced differentiation, decreased expression of myogenic genes, and increased the 27
expression of cell cycle-related genes, and the number of cells that remained in the cell cycle. 28
Knockdown of BAF180 had no effect on differentiation, suggesting that only the BRG1 and BRM 29
bromodomains contributed to differentiation. Comparison with existing gene expression data from 30
myoblasts subjected to knockdown of BRG1 or BRM showed that bromodomain function was 31
required for a subset of BRG1- and BRM-dependent gene expression. ChIP analysis revealed 32
decreased BRG1 and BRM binding to target gene promoters, indicating that the BRG1 and BRM 33
bromodomains promote chromatin binding. Thus mSWI/SNF ATPase bromodomains contribute 34
to cell cycle exit, to skeletal muscle-specific gene expression, and to stable promoter binding by 35
the mSWI/SNF ATPases. 36
37
INTRODUCTION 38
Regulation of gene expression is a tightly coordinated process that is dependent on transcription 39
factors, coactivators and chromatin remodelers. Some of these regulators are tissue-specific and 40
act on target genes in a context-dependent manner. Tissue-specific regulation is absolutely crucial 41
for proper development of multi-cellular life forms in which all cells contain the same genetic 42
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information. Portions of the genome that are irrelevant to a particular tissue type are often 43
condensed into repressive heterochromatin as development and differentiation occur (1, 2). In 44
contrast, coordinated activity of lineage-determining transcription factors and chromatin 45
remodelers, in particular the mSWI/SNF family of chromatin remodeling enzymes, drives many 46
differentiation events, including skeletal muscle differentiation (3–7). The mSWI/SNF enzymes 47
remodel chromatin in an ATP-dependent manner (8–10) and form a family of enzymes assembled 48
into different configurations from a potential pool of more than twenty subunit proteins (11–13). 49
The BRG1 and BRM ATPases act as mutually exclusive catalytic subunits (10). 50
Skeletal muscle originates from the paraxial mesoderm during embryogenesis. Fetal skeletal 51
myogenesis is characterized by an abundance of myogenic progenitor cells that divide actively and 52
fuse to form multinucleated muscle fibers (14, 15). As the embryo develops into an adult, these 53
progenitor cells become relatively sparse and quiescent. These adult stem cells are known as 54
satellite cells and can be activated to proliferate and regenerate new myofibers in case of an injury 55
to adult skeletal muscle (16–18). Upon activation, expression of myogenic regulatory factors 56
(MRFs) – MYOD, MRF4, MYF5 and Myogenin - is initiated in a coordinated manner. MRFs are 57
basic helix-loop-helix (bHLH) proteins that are evolutionarily conserved from worms to humans 58
(19–21). They bind to consensus sequences called E-boxes at target muscle promoters and activate 59
muscle-specific gene expression (22). Another family of transcription factors called the myocyte 60
enhancer factor 2 (MEF2) family acts with the MRFs to promote expression of the myogenic 61
genes (23, 24). 62
During skeletal myogenesis, the mSWI/SNF complex is recruited to the myogenic loci by MRFs 63
(25–30), in some cases, in conjunction with PBX1 (25). Mechanistically, upon induction of 64
differentiation in myocytes, the p38 kinase responds to extracellular cues by phosphorylating the 65
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BAF60c subunit of mSWI/SNF chromatin remodeling enzymes, which is associated with MYOD 66
on myogenic genes in the absence of other mSWI/SNF subunits in proliferating myoblasts (27, 67
29). The phospho-BAF60c-MYOD complex then recruits the rest of the mSWI/SNF complex to 68
myogenic loci, which promotes chromatin accessibility and activates gene expression (29). Once 69
recruited to myogenic loci, the ATPase activity of BRG1 or BRM in the complex is known to be 70
indispensable for expression of the differentiation-specific gene program (26, 28, 31). 71
The BRG1 and BRM ATPases possess bromodomains in the C-terminal part of the protein (32–72
34). Bromodomains are well-characterized motifs known to interact with acetylated lysine residues 73
on the N-terminal tails of histones H3 and H4 (35, 36) and on other non-histone proteins (37). The 74
interaction of bromodomains with acetylated histones has been determined to be crucial for 75
regulation of some gene expression events (37). Based on structural homology, bromodomain-76
containing proteins can be classified into eight families (35, 38). BRG1 and BRM belong to family 77
VIII of bromodomains along with a third mSWI/SNF protein called BAF180 (Polybromo1, 78
PBRM1, PB1) that contains six tandem bromodomains (38, 39) 79
In this study, we characterized the role of mSWI/SNF bromodomains in the context of skeletal 80
myogenesis. We showed that inhibiting bromodomain function using PFI-3, a specific 81
pharmacological inhibitor that binds to the BRG1, BRM and BAF180 bromodomains (40–42), 82
reduced the ability of mouse myoblasts to differentiate into myotubes. Using RNA-sequencing, 83
we identified the genes whose expression is dependent on mSWI/SNF bromodomains. Broadly, 84
proliferation-related genes were found to be upregulated by bromodomain inhibition while 85
myogenic genes were downregulated. We also demonstrated that bromodomain function is 86
essential for timely exit of myoblasts from the cell cycle upon induction of differentiation. We 87
determined that BAF180 is not required for myogenesis in mouse myoblasts and demonstrated 88
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that the BRG1 and BRM bromodomains play a crucial role in skeletal muscle differentiation by 89
promoting the stable binding of BRG1 and BRM to target gene promoters. Thus, this study 90
mechanistically demonstrates the specific importance of mSWI/SNF bromodomains in context of 91
skeletal muscle differentiation. 92
93
RESULTS 94
Inhibition of bromodomain function results in aberrant myotube fusion 95
PFI-3 is a pharmacological inhibitor specific for the BRG1, BRM and BAF180 bromodomains, 96
members of bromodomain family VIII (40, 41). Prior work showed that PFI-3 impaired 97
differentiation of immortalized pre-adipocytes and myoblasts (42). The mechanisms responsible 98
for the observed effects on differentiation were not defined, so we sought to investigate the roles 99
played by mSWI/SNF bromodomains during myogenesis. 100
C2C12 immortalized myoblasts and primary myoblasts isolated from the tibialis anterior muscles 101
of 1-week old C57BL/6 mice were assayed for their ability to differentiate in the presence of PFI-102
3 or the vehicle (DMSO). DMSO-treated C2C12 myoblasts immunostained for myosin heavy 103
chain (MHC) showed formation of longer and thicker myotubes at 48h and 72h post induction of 104
differentiation than did C2C12 myoblasts treated with PFI-3 (Fig 1a). The efficiency of myogenic 105
differentiation can be scored by calculating fusion index, which is the ratio of the number of nuclei 106
in MHC-stained cells to the total number of nuclei (43). C2C12 cells treated with PFI-3 showed a 107
>50% decrease in fusion index at 24h, 48h and 72h as compared to control samples (Fig 1b). 108
Similar results were observed when primary myoblasts were exposed to PFI-3. While DMSO-109
treated primary myoblasts showed elongated myotubes upon induction of differentiation, the PFI-110
3-treated cells showed fewer and less elongated myotubes at corresponding timepoints (Supp fig 111
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1a). Quantitative analysis of differentiated primary myoblasts immunostained for MHC showed 112
about a 25-30% decrease in fusion index (Supp fig 1b). 113
We further quantitatively analyzed the extent of differentiation by counting the number of nuclei 114
in MHC-positive myotubes and classifying them into groups at each timepoint. DMSO-treated 115
C2C12 cells shifted from the majority of 48h myotubes having <5 nuclei to the majority of 116
myotubes having >5 nuclei by 72h (Fig 1c). PFI-3-treated cells failed to make this switch; the 117
majority of 72h myotubes had <5 nuclei (Fig 1c). Similarly, in PFI-3 treated primary cells, the 118
number of myotubes with >5 nuclei at 36h was about one-third of those in DMSO-treated control 119
cells. In PFI-3 treated samples, cells with a single nucleus positively immunostained for MHC 120
were abundant, showing a failure of differentiating myoblasts to fuse (Supp fig 1c). These results 121
suggest that an initial myogenic stimulus is present but is not fully implemented due to inhibition 122
of bromodomain function. 123
Myogenic genes are downregulated upon PFI-3 induced inhibition of mSWI/SNF 124
bromodomains 125
The results show that PFI-3 treatment causes defects in myogenic differentiation, including an 126
inability of the differentiating myoblasts and/or nascent myotubes to fuse. Myomaker and 127
myomixer have been identified as master regulators of myoblast fusion (44–47). We therefore 128
determined whether the expression of these two regulators was altered upon PFI-3 induced 129
bromodomain inhibition. The results show that expression of these two genes was significantly 130
lower in PFI-3 treated C2C12 cells (Fig 2a). The expression of other myogenic genes like 131
myogenin, creatine kinase and myosin light chain 1 was also significantly decreased in PFI-3 132
treated samples, as was the expression of caveolin 3 and integrin 7A, two muscle differentiation-133
related genes (Fig 2b). Western blot analysis confirmed the decreased expression of myosin 134
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heavy chain in PFI-3 treated C2C12 cells (Fig 2c). Similar results were obtained for PFI-3 135
treated primary myoblasts (Supp fig 2). The gene expression signatures from both C2C12 cells 136
and primary myoblasts provides a molecular explanation for the differentiation phenotype caused 137
by bromodomain inhibition. 138
RNA-seq analysis of PFI-3 treated C2C12 cells shows upregulation of cell cycle genes and 139
downregulation of myogenic genes 140
To gain better insight into effect of the molecular mechanism of bromodomain inhibition on 141
skeletal muscle differentiation, we performed RNA-sequencing of C2C12 cells treated with 142
DMSO or PFI-3. Cells were harvested from proliferative stage (GM) and two differentiated stages 143
(DM 24h and DM 48h post-induction). Libraries generated from the samples had ~45M unique 144
reads. Transcripts were mapped to the mouse genome (mm10) and gene expression levels were 145
calculated. Genes that were identified to be differentially expressed in both replicates for each 146
condition and timepoint were considered for further analysis. 147
We first examined whether PFI-3 treatment affected gene expression of the subunits of mSWI/SNF 148
complexes. A recent characterization of sub-families of mSWI/SNF complexes identified 29 149
subunit proteins (13). Assessment of expression of the genes encoding each of these proteins at 150
each time point found only two instances of statistically significant differences (Supp. Table 1). 151
Arid1a expression was reduced ~7% at 24 h post-differentiation and Actl6a expression was 152
increased ~27% at 48 h post-differentiation. We conclude that PFI-3 treatment had essentially no 153
effect on the expression of the genes encoding mSWI/SNF subunits. 154
Inhibition of bromodomain function affected the expression of about 50% of the total genes 155
identified as expressed over the time course of the experiment (Fig 3a). The number of DEGs due 156
to bromodomain inhibition increased as a function of differentiation (Fig 3b). The total number of 157
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DEGs for proliferating cells (GM) and differentiating cells at 24h or 48h post-differentiation (DM 158
24h and DM 48h) were 3144 (up 2216; down 928), 4675 (up 2878; down 1797) and 5261 (up 159
3105; down 2156), respectively (Supp. Table 2). Gene expression at DM 24h and DM 48h was 160
strongly correlated with 2359 common DEGs as compared to about 1634 common DEGs between 161
GM and DM 24h. There were 899 genes that were differentially expressed at all timepoints. Gene 162
ontology (GO) analysis was performed on DEGs to cluster genes into function-based categories 163
(48, 49) and the complete results are listed in Supp. Table 3. GO analysis of genes downregulated 164
at DM 48h showed that the top 10 biological process categories were related to skeletal muscle 165
contraction and skeletal muscle tissue development (Fig 3c; Supp. Table 3). This is in agreement 166
with our experimental results, thus identifying the importance of bromodomain function in 167
myogenesis. The top 10 categories from GO analysis of genes upregulated at DM 48h were related 168
to cell proliferation (Fig 3d; Supp. Table 3), which indicated altered proliferation due to PFI-3-169
induced bromodomain inhibition. The promoters of the differentially expressed genes were also 170
analyzed using the HOMER motif enrichment software (49). Sequences 1kb upstream of the 171
transcription start sites were searched for presence of known consensus motifs (Supp. Table 4). 172
The analysis revealed that promoters of genes downregulated due to PFI-3 treatment were 173
significantly enriched in motifs corresponding to muscle specific transcription factors from the 174
MEF and MRF families. (Fig 3c; Supp. Table 4). In the case of upregulated genes, HOMER 175
analysis identified enrichment of motifs known to be bound by E2F family, NFY, KLF5 and Sp1 176
transcription factors (Fig 3d; Supp. Table 4). E2F and KLF5 TF families are known to play key 177
role in regulation of cell proliferation and differentiation (50–52). Thus, PFI-3 induced 178
bromodomain inhibition affects expression of genes which are involved in regulation of cell 179
proliferation and skeletal muscle differentiation. 180
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PFI-3 treatment blocked cell-cycle exit of C2C12 cells induced for differentiation 181
Cell cycle exit is prerequisite for cellular differentiation to proceed in a number of cell types (53–182
55). Results from the GO analysis of upregulated genes at DM 48h indicated that inhibition of 183
bromodomain function may interfere with cell cycle exit. To experimentally address the 184
requirement of mSWI/SNF bromodomain function in cell cycle exit, a BrdU (5-bromo-185
deoxyuridine) incorporation assay was performed with samples treated with or without PFI-3 (Fig 186
4a). Confocal microscopy analysis showed that cells treated with PFI-3 continued to incorporate 187
BrdU even after the control cells showed no further incorporation, indicating a partial inability to 188
exit cell cycle. Quantification of these images is shown in Fig 4b. Increased mRNA expression of 189
cyclin A2, cyclin B1, cyclin D1, and cyclin D2 in PFI-3 treated samples from DM 48h as compared 190
to DMSO controls further correlates with continued cell cycle (Fig 4c). These results show that 191
bromodomain inhibition allows some of the myoblasts to overcome the signals to exit cycle that 192
are normally provided by the low mitogen media and by contact inhibition. Thus, PFI-3 induced 193
bromodomain inhibition may be affecting two aspects of myogenesis: timely exit from the cell 194
cycle and the expression of myogenic genes. 195
BAF180 is dispensable for C2C12 myoblast differentiation 196
The composition of mSWI/SNF complexes is variable depending on function, cell-type and 197
context. Every functional mSWI/SNF complex contains either the BRG1 or the BRM ATPase, 198
while one major sub-class of mSWI/SNF complexes also contains BAF180 (10, 13). Thus, PFI-3 199
treatment affects all mSWI/SNF complexes. BRG1 and BRM have been shown to be required for 200
skeletal muscle differentiation (25, 26, 28, 30, 56, 57), but the requirement for BAF180 in this 201
process has not been evaluated. 202
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We knocked down BAF180 using siRNA. C2C12 cells depleted for BAF180 were induced for 203
differentiation alongside cells with scrambled siRNA treatment (Fig 5a, 5b). The cells showed no 204
phenotypic defect and differentiated normally. This result suggests that BAF180 may be 205
dispensable for myogenesis and implies that PFI-3 induced inhibition of myoblast differentiation 206
is mediated through inactivation of BRG1 and/or BRM bromodomain function. 207
Gene targets of PFI-3 inhibition of mSWI/SNF bromodomains predominantly overlap with 208
targets of BRG1 knockdown during myogenesis 209
The importance of BRG1 and BRM in skeletal muscle differentiation has been shown previously 210
by multiple groups. These studies have looked at muscle-specific gene expression profiles and 211
promoter binding of selected mSWI/SNF subunits on myogenic regulatory sequences. In a recently 212
published study, the authors performed siRNA-mediated knockdown of BRG1 in C2C12 cells 213
differentiated for 48h followed by RNA-sequencing analysis (58). We compared the siBRG1 214
dataset from this study with our RNA-sequencing dataset generated from PFI-3 treated C2C12 215
cells differentiated under similar conditions (Fig 6a). The rationale behind this comparison was to 216
get an understanding of the relative importance of the BRG1 bromodomain. The analysis showed 217
that 46% of the gene targets downregulated due to PFI-3 treatment overlapped with downregulated 218
genes in the siBRG1 dataset (Fig 6a). Similarly, 46% of the upregulated genes due to PFI-3 219
inhibition were common with upregulated genes from siBRG1 dataset. The results show that a 220
subset of BRG1-dependent gene expression in differentiating myoblasts requires bromodomain 221
function. GO analysis of the common overlapping genes was conducted. Common downregulated 222
genes belonged to muscle differentiation related processes while the common upregulated targets 223
fell into cell-cycle related categories. (Fig 6a-b; Supp. Table 5). Promoters of the common 224
upregulated and downregulated genes were also analyzed using HOMER to search for the presence 225
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of known consensus motifs within 1kb upstream of their TSS (Fig 6a-b; Supp. Table 5). Promoters 226
of upregulated genes contained motifs known to be bound by E2F, KLF, NFY and Sp1 TF families 227
while those of downregulated genes were enriched for motifs corresponding to MEF and MRF 228
muscle-specific transcription factor families (Fig 6a-b; Supp. Table 5). Thus, the results from the 229
GO and HOMER motif enrichment analyses of overlapping genes and their promoters are similar 230
to those from PFI-3 treatment as shown in Fig 3c-d and therefore point towards a crucial role 231
played by BRG1 bromodomain in skeletal muscle differentiation and cell cycle regulation. 232
We examined the genes that were dependent on BRG1 for expression but independent of PFI-3-233
mediated inhibiton of bromodomain function (Supp. Fig 3; Supp. Table 6). Genes that are up- and 234
down-regulated predominantly represented targets involved in metabolic processes and do not 235
include genes that control skeletal muscle differentiation or control of cell cycle. This suggests 236
that bromodomain-dependent regulation of gene expression is critical for myogenesis. This result 237
also is consistent with prior studies showing that ATPase domain function is required for BRG1-238
mediated regulation of metabolism (59, 60) and that PFI-3 treatment did not affect cancer cell 239
proliferation dependent on BRG1 and/or BRM (40, 60). 240
Microarray analysis of gene expression in C2C12 cells upon siRNA-mediated knockdown of 241
BRG1 or BRM has also been done in a prior study by Albini et. al (30). Despite the difference in 242
methodologies, we overlapped the DEGs from that study with our RNA-seq data from PFI-3 243
treated C2C12 cells at comparable timepoints (48h post-differentiation) to evaluate if the outcome 244
from this comparison is in consonance with the previous results. The analysis showed that more 245
than one-third of the genes identified by Albini et al. as unique BRG1 targets overlapped with 246
differentially expressed genes from our PFI-3 RNA-seq dataset (Supp Fig 4a). Upon looking 247
individually at upregulated and downregulated targets uniquely regulated by BRG1, there was a 248
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39% and 34% overlap, respectively (Supp Fig 4b). These common overlapping target genes were 249
then characterized using GO analysis (Supp Fig 4a-b). Upregulated genes fell into cell-cycle 250
related categories, and downregulated genes belonged to muscle differentiation related categories 251
(complete analysis in Supp. Table 7). This is in agreement with the overall results from PFI-3 252
treatment indicating the importance of active bromodomain for BRG1 function. Of the genes 253
identified by Albini et al. as unique BRM targets, only about 20% were common with PFI-3 DEGs 254
(Supp Fig 4b). These numbers show that more genes may be regulated by the BRG1 bromodomain 255
as compared to the BRM counterpart. Albini et al. also identified a set of genes that were 256
coregulated by both BRG1 and BRM. About 32% of these upregulated targets and 16% of the 257
downregulated targets were found in the corresponding list of differentially expressed genes due 258
to PFI-3 induced bromodomain inhibition (Supp Fig 4b; Supp. Table 7). This comparative analysis 259
further confirms that both BRG1 and BRM bromodomains play a role in skeletal myogenesis. 260
PFI-3 inhibition of mSWI/SNF bromodomains decreased binding of BRG1 and BRM to 261
target gene promoters. 262
BRG1-containing mSWI/SNF complexes are recruited to regulatory regions of myogenic genes 263
upon induction of muscle differentiation (25, 26, 29, 30, 61, 62). This step is required to induce 264
remodeling of chromatin at myogenic gene loci, thus allowing activation of muscle-specific gene 265
expression. 266
We performed ChIP experiments to determine whether the occupancy of BRG1 was affected at 267
the myogenic gene regulatory regions in response to bromodomain inhibition by PFI-3. As 268
expected, BRG1 occupancy at myogenic regulatory regions increased as a function of 269
differentiation in DMSO-treated samples (Fig 7a). However, BRG1 occupancy at the tested 270
regulatory sequences was partly inhibited in PFI-3 treated cells. This loss of binding correlates 271
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with the decreased expression of these genes as seen in previous results (Fig. 2). We also looked 272
at binding of BRG1 and BRM on cyclin D1 and cyclin D2 promoters. It is known that in C2C12 273
myoblasts differentiated for 48h, BRM binding to the cyclin D1 gene promoter is crucial for its 274
repression and cell-cycle exit (30). In that study, the authors showed that the expression of cyclin 275
D1 was co-regulated by both BRG1 and BRM during the later stages of differentiation (30). In 276
MCF-7 cells, BRG1 has been shown to bind to cyclin D1 promoter and regulate its expression 277
(63). In PFI-3 treated C2C12 cells, chromatin IPs at the cyclin D1 promoter showed a decrease in 278
occupancy of BRM and BRG1 (Fig 7b). Similarly, we saw a significant decrease in binding of 279
BRG1 and BRM at the cyclin D2 promoter due to PFI-3 treatment. These results show that the 280
bromodomain function of BRG1 and BRM contributes to their binding at target gene promoters. 281
282
DISCUSSION 283
Bromodomains in mSWI/SNF proteins 284
Bromodomains are a conserved structural motif found in only 46 human proteins, and they are 285
classified into eight families (38). Bromodomains bind to acetylated lysines, which facilitates 286
protein-protein interactions (37). The ability of bromodomains to target proteins to acetylated 287
nucleosomes containing acetylated histones has been predicted to be a mechanism by which 288
chromatin epigenetic modifications are read, thereby enabling translation of the histone mark via 289
the bromodomain protein or via proteins associated with the bromodomain-containing protein (37, 290
64, 65). Family VIII bromodomains include the 6 bromodomains found in the N-terminal portion 291
of the BAF180 protein, the bromodomains present in the BRG1 and BRM ATPases, and the 292
bromodomain found in the histone lysine N-methyltransferase ASH1L (38). 293
294
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Work to date on mSWI/SNF bromodomains has consisted of structural analyses (66–70), in vitro 295
studies of bromodomain binding to histones, DNA and nucleosomes (67, 70–74) and a limited 296
number of functional analyses in higher eukaryotes (75, 76). Of particular note, deletion of the 297
bromodomain in Drosophila BRM, which is the only SWI/SNF ATPase, had no effect on 298
developing or adult flies (77). In human cells, reconstitution of BRG1-deficient tumor cells with 299
wildtype or mutant versions of BRG1 determined that sequences C-terminal to the ATPase 300
domain, which includes the bromodomain, were not required for BRG1-mediated co-activation 301
of transcription by the glucocorticoid receptor (78). Similarly, BRG1 containing a bromodomain 302
mutation was capable of co-activating myocardin to promote smooth muscle-specific gene 303
expression (79). However, the BRG1 bromodomain directly contributes to the ability of 304
Repressor Element 1-silencing Transcription Factor (REST) to bind chromatin and repress target 305
genes (80). Mutation of each of the six BAF180 bromodomains revealed that four of the six 306
promoted tumor suppressor function, gene regulation, and chromatin affinity in clear cell renal 307
cell carcinoma cells (81). Other work showed that the 4th bromodomain of BAF180 mediated 308
interaction with acetylated p53, which promotes p53 binding to and transcriptional activation of 309
its target promoters (82). Thus, the requirement for functional mSWI/SNF protein 310
bromodomains is variable. It may be cell-type dependent as well. Here we demonstrate that the 311
BRG1 and/or BRM bromodomains, but not the BAF180 bromodomains, contribute to myogenic 312
differentiation. Both BRG1 and BRM are required for myogenic differentiation; this work is the 313
first to identify a contribution to myogenesis by any domain other than the ATPase domain. The 314
work further supports the idea of context-dependent requirements for mSWI/SNF bromodomain 315
functions. 316
317
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Use of PFI-3 to probe biological function of mSWI/SNF subunits containing bromodomains 318
Efforts to identify pharmacological inhibitors of bromodomains identified salicylic acid as a 319
specific interactor of BRG1, BRM, and BAF180 bromodomains (41, 83). This led to a series of 320
structure-guided design steps that resulted in the PFI-3 inhibitor that is specific for the second and 321
fifth bromodomain of BAF180 and the bromodomains of BRG1 and BRM (41, 42). 322
323
To date PFI-3 has been used to probe biological function in a number of contexts. Because BRG1 324
and other subunits of the mSWI/SNF enzyme have been shown to be required for proliferation of 325
some cancer cells (84–86), PFI-3 was tested for inhibitory effects on cancer cell proliferation, with 326
no effect observed on many different cancer cell types, including the NCI-60 tumor cell panel (40, 327
41, 87). These results demonstrate that mSWI/SNF bromodomains are not required for cancer cell 328
proliferation. An inhibitory effect of PFI-3 was observed in PTEN-depleted prostate cancer cells 329
in culture, in xenografts and in PTEN deficient mouse model susceptible to prostate cancer (88). 330
PTEN is a tumor suppressor that normally regulates the AKT/PKB signaling pathway (89). Thus, 331
the importance of mSWI/SNF bromodomains, and specifically, the BRG1 bromodomain, is 332
enhanced in the absence of PTEN and inhibition of AKT/PKB signaling, suggesting a complex 333
mode of regulation of mSWI/SNF protein bromodomain function. PFI-3 treatment increased the 334
accessibility of an mSWI/SNF-repressed promoter and its gene expression (90). In these and other 335
studies, PFI-3 altered both gene expression patterns dependent on mSWI/SNF bromodomain-336
containing proteins and the cellular and organismal phenotypes controlled by those genes (87, 88, 337
91–96). The data indicate that the pleiotropic effects of PFI-3 and inhibition of mSWI/SNF 338
bromodomains links to the ability of the chromatin remodeling enzyme to modulate gene 339
expression. 340
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16
341
In the realm of tissue specification, PFI-3 treatment caused a loss of “stemness” and promoted 342
differentiation of ESCs and trophoblast and neural stem cells in the absence of differentiation 343
signaling (41, 97). In other contexts, PFI-3 inhibited differentiation, blocking the ability of 344
myoblasts and pre-adipocytes to form myotubes and adipocytes, respectively, in the presence of 345
differentiation signaling (42). In this report, we investigated the mechanisms responsible for the 346
inhibitory effects of PFI-3 on myogenesis. PFI-3 treatment affected bromodomain function of 347
BRG1 and BRM, but BAF180, as BAF180 was dispensable for differentiation. BRG1/BRM 348
bromodomain function was required for appropriate regulation of cell cycle withdrawal and 349
initiation of the myogenic gene expression program, with mis-regulation of a subset of the genes 350
regulated by BRG1 and BRM. Deficient gene regulation was linked to the partial inhibition of the 351
ability of BRG1 and BRM to bind to target gene regulatory sequences, reflecting a necessary 352
contribution of these bromodomains to promote interaction of the mSWI/SNF enzymes with 353
chromatin. 354
355
BAF180 is dispensable for myogenesis 356
mSWI/SNF complexes are a family of enzyme complexes marked by diversity of subunit 357
composition (6, 11, 98). Initial descriptions of mSWI/SNF complexes reported two separable 358
biochemical fractions that showed ATP-dependent chromatin remodeling activity (8–10). These 359
complexes have become known as BAF (BRG1/BRM-associated factors) and PBAF 360
(Polybromo-associated BAF), the latter taking its name from the presence of the BAF180 protein 361
that is specific to this complex. However, both BAF and PBAF complexes themselves are merely 362
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17
separable groups of complexes that contain both shared and unique subunits (99, 100). A third 363
family of complexes, called ncBAF (noncanonical BAF) was identified more recently (13, 101). 364
365
Although there are many subtypes of functional mSWI/SNF complexes in the cell, all of them 366
necessarily have at least one of the proteins from the family VIII of bromodomains (102). BAF 367
and ncBAF complexes contain either BRG1 or BRM, while PBAF complexes contain BRG1 and 368
BAF180 (13). BAF180 has been implicated in DNA damage repair (103, 104) and is also 369
required for cardiac development (105, 106); knockout in mice caused severe hypoplastic 370
ventricle development and trophoblast placental defects (105). However, adult mice with 371
BAF180 depletion were phenotypically normal except for a hematopoietic stem cell defect 372
observed in aged mice (107). Similarly, BAF180 shows tumor-suppressive properties in some 373
but not all cancer cell lines (107–111). These findings are consistent with context-specific 374
requirements for the BAF180 protein. 375
376
In our work, we used siRNA-mediated knockdown to show that BAF180 is dispensable for 377
skeletal muscle differentiation. This suggests that the PBAF family of mSWI/SNF complexes are 378
also dispensable in this differentiation program. Although there are many reports characterizing 379
the requirement for mSWI/SNF complexes in myogenesis, focus has been limited to the two 380
ATPase subunits, to BAF47/INI1, which is shared by BAF and PBAF complexes, and to the 381
BAF60 subunit that is shared by all subfamilies of mSWI/SNF complexes (25–27, 29–31, 56, 57, 382
112–125). A prior report documented the binding of BAF250A to myogenic promoters (115), 383
perhaps implicating BAF complexes as the relevant mSWI/SNF enzyme subfamily for myogenic 384
differentiation, but the requirement for BAF250A was not evaluated. Nevertheless, a requirement 385
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for specialized complexes for specific gene regulation events is one of the main hypotheses for 386
existence of diverse families of mSWI/SNF complexes. 387
388
BRG1 and BRM bromodomain function contribute to the regulation of myogenic 389
differentiation 390
BRG1 and BRM contribute to the activation of the myogenic gene expression program and BRM 391
contributes to the cell cycle arrest of myoblasts that precedes differentiation (25–30, 56). Inhibition 392
of the mSWI/SNF bromodomains by PFI-3 recapitulated these findings, indicating that the 393
bromodomains of BRG1 and BRM are needed to both regulate cell cycle exit and for the initiation 394
of tissue-specific gene expression. RNA-seq analysis of PFI-3 treated cells provided evidence of 395
global disruption of the regulation of cell cycle exit and the initiation of myogenic gene expression. 396
A recently published RNA-seq study investigating the role of chromatin remodeling in skeletal 397
myogenesis performed knockdown of BRG1 and evaluated gene expression at timepoints 398
comparable with our study (58). Comparison of this dataset with ours identified a large overlapping 399
subset of gene targets involved in cell cycle exit and myogenesis indicating that BRG1 400
bromodomain plays a crucial role in regulation of BRG1-dependent events in skeletal muscle 401
differentiation. 402
Additionally, prior microarray-based studies of myogenic gene expression upon BRG1 and BRM 403
knockdown also identified these processes as being BRG1- and BRM-dependent (30). Despite the 404
differences in platforms, we integrated the two datasets. There was a considerably greater overlap 405
between PFI-3 affected genes and genes mis-regulated by BRG1 knockdown than there was 406
between PFI-3 affected genes and genes mis-regulated by BRM knockdown. This may suggest 407
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19
that a greater percentage of genes that require BRG1 are also dependent on the BRG1 408
bromodomain than is true for the set of genes that require BRM. 409
410
Regardless, our ChIP experiments demonstrated that bromodomain inhibition resulted in a 411
decreased ability of BRG1 to bind to genes activated during the myogenic differentiation protocol 412
and a decreased ability of BRG1 and BRM to bind to genes controlling cell cycle. The chromatin 413
interacting properties of the BRG1 and BRM bromodomains therefore likely contribute to gene 414
expression. The principles determining the variable requirements for BRG1, BRM and BAF180 415
bromodomains in different cellular contexts remain to be investigated. However, many of the 416
mSWI/SNF subunits have additional domains that could promote chromatin interaction. For 417
instance, BRG1 and BRM proteins have AT hooks, BAF180 and the BAF57 protein have an HMG 418
box-like domain, the ARID1A/1B and ARID2 proteins have ARID domains that may mediate 419
nucleic acid interactions, and BRD7 and BRD9 also have bromodomains. The requirement for any 420
of these domains may be dependent on the protein makeup of the particular mSWI/SNF complex 421
and the presence or absence of these other domains. Such a scenario suggests that these largely 422
non-sequence specific DNA-binding domains function in an additive or cooperative manner to 423
help facilitate chromatin interactions and remodeling events. Additional characterization of the 424
putative chromatin interactions domains in the context of differentiation will be necessary to 425
determine whether they are required and act in concert to promote mSWI/SNF interaction with 426
chromatin and function. 427
428
MATERIALS AND METHODS 429
Antibodies and Chemicals 430
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Antibodies were purchased from Santa Cruz Biotech, USA (anti-BRG1, sc-17796; anti-BrdU, sc-431
32323). Myosin Heavy chain antibody (#MF20) was purchased from the Developmental Studies 432
Hybridoma Bank, University of Iowa, USA. BRM antisera was described previously (126). Lysis 433
buffers for ChIP assays were purchased from Cell Signaling Technology, USA (SimpleChIP® 434
Enzymatic Cell Lysis Buffers A & B, 14282; SimpleChIP® Chromatin IP Buffers, 14231). 435
Dulbecco’s modified Eagle's medium (DMEM) was purchased from ThermoFisher Scientific 436
(#11965118). 437
Cell culture 438
C2C12 cells were purchased from ATCC (Manassas, VA) and maintained at sub-confluent 439
densities in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 440
incubator at 37°C in 5% CO2. 441
Mouse satellite cells were isolated from leg muscles of 2 week old C57BL6/J mice using Percoll 442
sedimentation followed by differential plating as described previously (114). Mice were housed in 443
the animal care facility at the University of Massachusetts Medical School and used in accordance 444
with a protocol approved by the Institutional Animal Care and Use Committee (IACUC). 445
For differentiation, cells at 70% confluency were switched to DMEM medium supplemented 446
with 2% horse serum and 2 g/ml of bovine insulin (Sigma-Aldrich, St. Louis, MO). Where 447
indicated, cells were pre-treated with DMSO or PFI-3 (Cayman Chemical, Ann Arbor, MI) for 448
24h before inducing differentiation. PFI-3 was maintained during the course of the experiment at 449
50M and the medium was replaced every 24 hours. 450
siRNA transfection 451
C2C12 cells were plated on 24-well plates in DMEM medium 24h before transfection. Cells were 452
transfected at 30-40% confluence using the Lipofectamine 2000 (Invitrogen) reagent with 50 nM 453
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siRNA SMARTpool ON-TARGETplus (Dharmacon, Scrambled non-targeting Pool # D-001810-454
10-20 and Pbrm1 #L-044781-00-0005). 48h post-transfection, the cells were induced for 455
differentiation and samples were harvested at indicated times for further analysis. 456
Immunocytochemistry 457
Cells were seeded on 22mm x 22mm size coverslips in 35mm dishes and were harvested after the 458
indicated treatments at the specified timepoints. The samples were washed with PBS twice and 459
then fixed with ice-cold fixative (2% formaldehyde and 0.2% glutaraldehyde in PBS) for 10 460
minutes on ice. The cells were washed with PBS twice and permeabilized with ice-cold 461
permeabilization buffer (0.2% Triton-X 100 in PBS) for 5 minutes on ice. Samples were then 462
washed once with PBS and blocked using freshly prepared blocking reagent (5% BSA in PBS) for 463
30 minutes at room temperature (RT). The cells were washed twice with PBS and incubated with 464
primary antibody cocktail diluted to the desired concentration in blocking reagent (2% BSA in 465
PBS) for 2 hours at room temperature. Post-incubation, samples were washed thrice with PBS 10 466
minutes each to remove non-specific binding. Cells were then incubated with fluorophore-467
conjugated secondary antibody (1:100) and DAPI (2g/ml), both diluted in blocking reagent (2% 468
BSA in PBS) for 45 minutes at RT followed by 3 washes with PBS to remove non-specific staining. 469
The stained cells on coverslips were then inverted-mounted on glass slides in 70% glycerol and 470
the sides were sealed with nail paint. Confocal imaging analysis was performed using Leica TCS 471
SP5 II laser scanning confocal microscope and analyzed with Leica Lite software. 472
Fusion Index 473
For calculation of fusion index, cells were harvested at the specified timepoints after the indicated 474
treatments and were immunostained with myosin heavy chain (MF20, DSHB) and DAPI (nuclear 475
staining) as described above. The images were captured at 40X magnification using a Leica TCS 476
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SP5 II laser scanning confocal microscope. Analysis was performed by scoring cells for number 477
of nuclei and MHC staining. Fusion index was calculated as the percentage of nuclei/cells stained 478
with myosin heavy chain as compared to total number of nuclei/cells (43). 479
RIPA buffer Lysis 480
Cells were harvested after the indicated treatments at specific timepoints and were washed twice 481
with ice-cold PBS. After draining all residual PBS, cells were scraped into 1ml ice-cold PBS with 482
1X protease inhibitor cocktail (Sigma Aldrich, P8340) and pelleted at 1500 X g for 5 minutes at 483
4C. The pellets were lysed in 500ul RIPA buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1mM 484
EDTA, 1% NP-40 and 0.25% sodium deoxycholate) supplemented with 1X protease inhibitor 485
cocktail (Sigma Aldrich, P8340). Samples were incubated on ice for 30 minutes and whole cell 486
extracts were prepared by passing the lysed pellets through a 27-gauge needle at least 4-5 times. 487
Samples were centrifuged at 14000 X g for 10 minutes at 4C and supernatants were collected. 488
Western Blot Analysis 489
Protein concentrations were determined using a PierceTM BCA protein assay kit (ThermoFisher 490
Scientific, USA) according to the manufacturer’s protocol. Equal amounts of protein from each 491
sample were aliquoted and mixed with 4X Laemmli Sample Buffer (BioRad) and boiled at 95C 492
for 10 minutes. The samples were electrophoresed on denaturing SDS-polyacrylamide gels and 493
transferred onto Immobilon-P PVDF membranes (Merck Millipore, USA). The membranes were 494
then blocked using 5% non-fat milk in PBS for 30 minutes followed by overnight incubation at 495
4C with primary antibody against protein of interest at the desired dilution in 2% non-fat milk 496
prepared in PBS. This was followed by 3 washes with TBS containing 0.1% Tween-20 for 5 497
minutes each at room temperature. The membranes were then incubated with HRP-conjugated 498
anti-mouse or anti-rabbit secondary antibodies (1:2500, GE Healthcare Life Sciences) diluted in 499
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2% non-fat milk prepared in PBS for 1 hour at RT followed by 3 washes with TBS containing 500
0.1% Tween-20 for 5 minutes each at room temperature. Chemiluminescent detection was 501
performed with ECL Plus (GE Healthcare Life Sciences) using an Amersham Imager 600 (GE 502
Healthcare Life Sciences). Representative blots from 3 independent experiments are shown. Band 503
signal intensities were quantified using ImageJ software (NIH) (127). 504
RNA isolation and quantitative RT-PCR 505
For RNA isolation, cells were grown in 35mm dishes and harvested after the indicated treatments 506
at specified timepoints. The media were removed, and cells were washed twice with PBS before 507
adding 1ml of Trizol (ThermoFisher) to each sample. RNA extraction was performed as per the 508
manufacturer’s protocol. The final RNA pellet was resuspended in 50l nuclease-free water. RNA 509
concentrations were quantified using a Nanodrop1000 spectrophotometer (ThermoFisher 510
Scientific). cDNA was prepared using 2g of total RNA using Superscript III First Strand 511
Synthesis Kit (Invitrogen) according to manufacturer’s protocol. 512
For qRT-PCR, 15l reactions were prepared in duplicate for all desired samples using 1l each of 513
forward and reverse primers (10M stocks) and 2l cDNA template, and the volume was brought 514
to 7.5l using UltraPure distilled water. 7.5l of Fast SYBR Green 2X Master Mix (Applied 515
Biosystems) was added to the reaction. The samples were run using the default protocol in 516
QuantStudio 3 RT-PCR machine (Applied Biosystems). Fold-changes were calculated using the 517
2−ΔΔCt method (128). Primer sequences are listed in Supp. Table 8. 518
RNA-sequencing analysis 519
For RNA sequencing, RNA samples were prepared as described above. Duplicate samples for each 520
timepoint were evaluated for quality and concentration at the UMass Medical School MBCL 521
Fragment Analyzer services. QC-approved samples were sent to BGI Americas Corporation for 522
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library preparation and RNA sequencing (129). Libraries were sequenced using the BGISEQ-500 523
platform and reads were filtered to remove adaptor-polluted, low quality and high content of 524
unknown base reads. About 99% of the raw reads were identified as clean reads (~65M). Of these, 525
about 70% of the reads were uniquely mapped to mouse reference genome mm10 using HISAT 526
(130). Transcripts were reconstructed using StringTie (131), and novel transcripts were identified 527
using Cufflinks (132). All transcripts were then combined and mapped to the mm10 reference 528
transcriptome using Bowtie2 (133). Gene expression levels were calculated using RSEM (134). 529
DEseq2 (135) and PoissonDis (136) algorithms were used to detect differentially expressed genes 530
(DEG). GO analysis was performed on DEGs to cluster genes into function-based and pathway-531
based categories (48, 49). Motif analysis was performed using HOMER motif discovery software 532
as described previously (49). For each differentially expressed gene, motif enrichment analysis 533
was performed at promoters for locations up to 1 kb upstream of the TSS. 534
BrdU incorporation and immunostaining 535
Cell were grown on coverslips in 35mm dishes with the indicated treatments. The culture media 536
was replaced with fresh media containing 10mM BrdU 30 minutes prior to harvesting to allow the 537
cells in S-phase to incorporate the nucleoside analog. Harvested samples were fixed and 538
permeabilized as described above for immunocytochemistry. For DNA hydrolysis, samples were 539
then incubated with 1.2N HCl diluted in PBS for 1 hour at 37°C, followed by neutralization in 540
0.1M sodium borate for 5 minutes at RT. The samples were washed with PBS thrice and 541
immunostained as mentioned above. 542
Chromatin Immunoprecipitation assay 543
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Chromatin immunoprecipitation assays were performed as described previously (115). 544
Quantification was performed using the fold enrichment method (2-(Ct sample – Ct IgG)) and shown as 545
relative to a control region. Sequences of primers used for ChIP assays are listed in Supp. Table 8. 546
Statistical analysis 547
All quantitative data for gene expression and chromatin immunoprecipitation are shown as mean 548
+/- the standard deviation of at least three independent biological replicates. In the fusion index 549
and BrdU incorporation assays, two independent experiments were performed in duplicate. A 550
minimum of 200 nuclei per sample were counted and the results were expressed as the mean of 551
calculated fusion index +/- standard deviation. Statistical analyses were performed using Graphpad 552
Prism8 Student’s t-test with two-tailed distribution and equal variance (Graphpad Prism Software 553
Inc.). Significance is displayed with *p<0.05, **p<0.01 and ***p<0.005. 554
Data Availability 555
The data from this RNA-seq study has been deposited in NCBI’s Gene Expression Omnibus and 556
are accessible through GEO accession number GSE151218. 557
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ACKNOWLEDGEMENTS 558
We thank T Padilla-Benavides, S Syed, and J Dilworth for comments and suggestions. This work 559
was funded by NIH grants GM56244 and GM136393 to ANI. 560
561
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27
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45
Figure Legends: 959
960
Fig. 1 (a) Confocal (top, scale 5μm) and bright field (bottom, scale 20μm) images for C2C12 961
myoblasts treated with DMSO or PFI-3 and stained for myosin heavy chain (green) and with 962
DAPI (blue) at the indicated timepoints. (b) Quantification of fusion index. (c) Differentiated 963
myoblasts at respective timepoints were analyzed for number of nuclei per myotube. *p<0.05, 964
**p<0.01 and ***p<0.005 965
966
Fig. 2 mRNA expression levels at the indicated timepoints for (a) the fusion regulator genes 967
myomaker and myomixer and (b) the myogenic genes myogenin, muscle creatine kinase, myosin 968
light chain 1, Caveolin 3 and Integrin 7α in C2C12 myoblasts treated with DMSO or PFI-3. 969
Expression was normalized to a control gene (EEF1A1). 100% expression is defined as the 970
timepoint at which maximal expression was observed. ns, not significant, *p<0.05, **p<0.01 and 971
***p<0.005. (c) Representative western blot for MHC expression at the indicated times in 972
C2C12 cells treated with DMSO or PFI-3. The indicated numbers are the pixel counts 973
normalized to β-tubulin expression calculated using ImageJ. 974
975
Fig. 3 (a) Heat maps showing results from RNA-seq analysis of PFI-3 treated C2C12 cells 976
assayed while in the proliferative stage in growth media (GM) and while in differentiation media 977
(DM) for 24h and 48h. (b) Venn diagram showing the number of genes affected at different 978
timepoints. There were 572 differentially expressed genes (DEGs) in GM (blue), 1319 DEGs in 979
DM 24h (peach) and 1681 DEGs in DM 48h (green). (c) GO analysis of downregulated genes at 980
48h post induction of differentiation shows downregulation of muscle related genes. A HOMER 981
.CC-BY-NC 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
46
motif search shows enrichment of motifs corresponding to muscle-specific transcription factor 982
families. (d) GO analysis of upregulated genes at 48h post induction of differentiation shows 983
upregulation of cell cycle related processes. A HOMER motif search shows enrichment of 984
transcription factor motifs associated with cell cycle regulators. 985
986
Fig. 4 (a) C2C12 cells treated with PFI-3 show continued BrdU incorporation after 36h and 48 h 987
post-induction of differentiation as compared to control cells, scale 5 μm. (b) Quantification of 988
confocal images for BrdU incorporation assay in DMSO or PFI-3 treated C2C12 cells at the 989
indicated timepoints. (c) mRNA expression levels of cyclin A2, cyclin B1, cyclin D1, and cyclin 990
D2 in C2C12 myoblasts treated with DMSO or PFI-3 for indicated timepoints. Expression was 991
normalized to a control gene (EEF1A1). 100% expression is defined as the timepoint at which 992
maximal expression was observed. ns, not significant, *p<0.05, **p<0.01 and ***p<0.005 993
994
Fig. 5 (a) Western blot analysis showed siRNA-mediated silencing of endogenous BAF180 in 995
proliferating C2C12 cells. A scramble siRNA (siScr) was used as a control. The indicated 996
numbers are the pixel counts normalized to Laminβ expression calculated using ImageJ. (b) 997
Representative images of myosin heavy chain staining in 48h and 96h differentiated cells 998
transfected with the control or BAF180-targeting siRNAs. The cells were fixed and analyzed by 999
immunofluorescence using an anti-myosin heavy chain mAb MF20 (green). The nuclei were 1000
visualized by DAPI staining (blue). Scale bar, 20 µm. 1001
1002
Fig. 6 Comparative analysis of RNA-seq datasets from BRG1 knockdown performed by Zhu et 1003
al. (NAR, 2020) and from PFI-3 treatment. Venn diagrams represent DEGs in corresponding 1004
.CC-BY-NC 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
47
datasets. The overlap represents genes common to both datasets. GO and HOMER motif 1005
enrichment analyses show biological process categories and motifs identified within 1kb 1006
upstream of the TSS in the promoters of (a) common downregulated genes and (b) common 1007
upregulated genes. 1008
1009
Fig. 7 ChIP assays show decreased occupancy of BRG1 and BRM on regulatory regions of 1010
target genes upon PFI-3 inhibition. (a) Bar plots for BRG1 occupancy on the myogenin promoter 1011
(Myog P), myosin heavy chain promoter (MHCIIb P), creatine kinase promoter (Ckm P) and 1012
creatine kinase enhancer (Ckm E) are shown. (b) Bar plots for BRG1 and BRM occupancies on 1013
the cyclin D1 and cyclin D2 promoters. The values have been normalized to an IgG experimental 1014
control. These values are also normalized for binding at a non-specific region. ns, not significant, 1015
*p<0.05, **p<0.01 and ***p<0.005 1016
1017
Fig. 8 Graphical summary showing the effects of PFI-3 induced bromodomain inhibition on 1018
skeletal myogenesis. In normal conditions, BRG1/BRM with active bromodomains can bind to 1019
promoters of target genes when muscle differentiation is induced. This in turn affects two 1020
important aspects of skeletal myogenesis: cell cycle exit and the formation of differentiated 1021
multinucleated myotubes. In the presence of PFI-3, BRG1 and BRM show reduced binding to 1022
target gene promoters leading to continued cell-cycle and incomplete differentiation resulting in 1023
shorter myotubes. 1024
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The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
Fig. 1 (a) Confocal (top, scale 5μm) and bright field (bottom, scale 20μm) images for C2C12 myoblasts treated with DMSO or
PFI-3 and stained for myosin heavy chain (green) and with DAPI (blue) at the indicated timepoints. (b) Quantification of fusion
index. (c) Differentiated myoblasts at respective timepoints were analyzed for number of nuclei per myotube. *p<0.05, **p<0.01
and ***p<0.005
a b
c
DM
SO
PF
I-3
DM
SO
PF
I-3
0h 24h 48h 72h
0h 24h 48h 72h0
20
40
60
80
Fusion Index
MH
C p
ositi
ve c
ells
(%
) DMSO
PFI-3
✱✱✱
✱✱✱
✱✱
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150
Nuclei per myotube
MH
C p
ositi
ve c
ells
with
indic
ate
d n
um
ber
of nucle
i (%
)
<5
>5✱✱✱
✱✱✱
.CC-BY-NC 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
Fig. 2 mRNA expression levels at the indicated timepoints for (a) the fusion regulator genes myomaker and myomixer and (b)
the myogenic genes myogenin, muscle creatine kinase, myosin light chain 1, Caveolin 3 and Integrin 7α in C2C12 myoblasts
treated with DMSO or PFI-3. Expression was normalized to a control gene (EEF1A1). 100% expression is defined as the
timepoint at which maximal expression was observed. ns, not significant, *p<0.05, **p<0.01 and ***p<0.005. (c) Representative
western blot for MHC expression at the indicated times in C2C12 cells treated with DMSO or PFI-3. The indicated numbers are
the pixel counts normalized to β-tubulin expression calculated using ImageJ.
a b
c
0h D
MSO
0h P
FI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150Myomaker
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
✱✱✱
✱✱✱
0h D
MSO
0h P
FI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150Myomixer
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
ns
✱✱✱
0h D
MSO
0h P
FI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150Myogenin
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
✱
✱✱✱ ✱✱✱
0h D
MSO
0h P
FI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150Creatine Kinase
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
ns
✱✱✱
✱✱✱
0h D
MSO
0h P
FI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150Myosin Light Chain 1
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
✱✱✱
✱✱✱
0h D
MSO
0h P
FI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150Caveolin 3
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
✱✱✱
✱
0h D
MSO
0h P
FI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
72h
DM
SO
72h
PFI-3
0
50
100
150Integrin 7α
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
ns
✱✱✱
MHC
β-tubulin
GM
DM
SO
48
h D
MS
O
48
hP
FI-
3
0h
DM
SO
0h
PF
I-3
GM
PF
I-3
24
h D
MS
O
24
h P
FI-
3
200kDa -
50kDa -
1.56 0.34
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The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
a
b
c
d
DM 48h Downregulated
DM 48h Upregulated
TF DNA Motif
NFY
E2F4
E2F6
KLF14
E2F1
E2F
ISRE
E2F7
IRF2
Sp1
TF DNA Motif
Mef2d
Mef2c
Myog
Mef2a
Ascl1
E2A
Mef2b
Ptff1a
Tcf12
Jun-AP1
899
735 545
1460
965
1581 2357
GM
24h 48h
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Fig. 3 (a) Heat maps showing results from RNA-seq analysis of PFI-3 treated C2C12 cells assayed while in the proliferative
stage in growth media (GM) and while in differentiation media (DM) for 24h and 48h. (b) Venn diagram showing the number of
genes affected at different timepoints. There were 3144 differentially expressed genes (DEGs) in GM (blue), 4674 DEGs in DM
24h (peach) and 5261 DEGs in DM 48h (green). (c) GO analysis of downregulated genes at 48h post induction of differentiation
shows downregulation of muscle related genes. A HOMER motif search shows enrichment of motifs corresponding to muscle-
specific transcription factor families. (d) GO analysis of upregulated genes at 48h post induction of differentiation shows
upregulation of cell cycle related processes. A HOMER motif search shows enrichment of transcription factor motifs associated
with cell cycle regulators.
.CC-BY-NC 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
Fig. 4 (a) C2C12 cells treated with PFI-3 show continued BrdU incorporation after 36h and 48h post-induction of differentiation as
compared to control cells, scale 5 μm. (b) Quantification of confocal images for BrdU incorporation assay in DMSO or PFI-3 treated
C2C12 cells at the indicated timepoints. (c) mRNA expression levels of cyclin A2, cyclin B1, cyclin D1, and cyclin D2 in C2C12
myoblasts treated with DMSO or PFI-3 for indicated timepoints. Expression was normalized to a control gene (EEF1A1). 100%
expression is defined as the timepoint at which maximal expression was observed. ns, not significant, *p<0.05, **p<0.01 and
***p<0.005
a b
c
GM 0h 12
h24
h36
h48
h
0
20
40
60
80
100
Brd
U p
ositi
ve (
%)
DMSO
PFI-3
✱✱ ✱✱
GM
DM
SO
GM
PFI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
0
50
100
150Cyclin D1
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
ns✱
GM
DM
SO
GM
PFI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
0
50
100
150Cyclin B1
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
✱✱✱
✱✱✱
GM
DM
SO
GM
PFI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
0
50
100
150Cyclin A2
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
ns
✱✱✱
GM
DM
SO
GM
PFI-3
24h
DM
SO
24h
PFI-3
48h
DM
SO
48h
PFI-3
0
50
100
150
mR
NA
expre
ssio
n r
ela
tive to
contr
ol g
ene (EEF1A1)
Cyclin D2
✱✱✱
✱✱✱
.CC-BY-NC 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
Fig. 5 (a) Western blot analysis showed siRNA-mediated silencing of endogenous BAF180 in proliferating C2C12 cells. A
scramble siRNA (siScr) was used as a control. The indicated numbers are the pixel counts normalized to Laminβ expression
calculated using ImageJ. (b) Representative images of myosin heavy chain staining in 48h and 96h differentiated cells
transfected with the control or BAF180-targeting siRNAs. The cells were fixed and analyzed by immunofluorescence using an
anti-myosin heavy chain mAb MF20 (green). The nuclei were visualized by DAPI staining (blue). Scale bar, 20 µm.
a
b 96h DM48h DM
siS
crsi
BA
F1
80
BAF180
Laminβ
WTscr BAF180
siRNA
70kDa -
200kDa -
0.88 1.19 0.15
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The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
DM 48h Downregulated DM 48h Upregulateda b
Fig. 6 Comparative analysis of RNA-seq datasets from BRG1 knockdown performed by Zhu et al. (NAR, 2020) and from PFI-3
treatment. Venn diagrams represent DEGs in corresponding datasets. The overlap represents genes common to both datasets.
GO and HOMER motif enrichment analyses show biological process categories and motifs identified within 1kb upstream of the
TSS in the promoters of (a) common downregulated genes and (b) common upregulated genes.
siBRG1 46% PFI-3siBRG1 46% PFI-3TF DNA Motif
E2F4
NFY
E2F1
E2F7
E2F6
E2F
KLF14
KLF5
Sp1
CHR
TF DNA Motif
Mef2d
Mef2c
Mef2b
Mef2a
Myog
Myf5
E2A
Smad4
ZNF322
Ap4
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The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
Fig. 7 ChIP assays show decreased occupancy of BRG1 and BRM on regulatory regions of target genes upon PFI-3 inhibition.
(a) Bar plots for BRG1 occupancy on the myogenin promoter (Myog P), myosin heavy chain promoter (MHCIIb P), creatine
kinase promoter (Ckm P) and creatine kinase enhancer (Ckm E) are shown. (b) Bar plots for BRG1 and BRM occupancies on the
cyclin D1 and cyclin D2 promoters. The values have been normalized to an IgG experimental control. These values are also
normalized for binding at a non-specific region. ns, not significant, *p<0.05, **p<0.01 and ***p<0.005
ab
GM
DM
SO
GM
PFI-3
48h
DM
SO
48h
PFI-3
0
5
10
15
20
Myog P
Fold
enrichm
ent/C
ontr
ol r
egio
n
BRG1
ns
✱✱✱
GM
DM
SO
GM
PFI-3
48h
DM
SO
48h
PFI-3
0
10
20
30
40
MHCIIb P
Fold
enrichm
ent/C
ontr
ol r
egio
n
BRG1
ns
✱
GM
DM
SO
GM
PFI-3
48h
DM
SO
48h
PFI-3
0
20
40
60
Ckm P
Fold
enrichm
ent/C
ontr
ol r
egio
n
BRG1
ns
✱✱✱
GM
DM
SO
GM
PFI-3
48h
DM
SO
48h
PFI-3
0
5
10
15
20
Ckm E
Fold
enrichm
ent/C
ontr
ol r
egio
n
BRG1ns
✱✱✱
GM
DM
SO
GM
PFI-3
48h
DM
SO
48h
PFI-3
0
2
4
6
8
CyclinD1 P
Fold
enrichm
ent/Ig
G/C
ontr
ol r
egio
n
BRG1
BRM
ns
ns
✱✱✱
✱✱
GM
DM
SO
GM
PFI-3
48h
DM
SO
48h
PFI-3
0.0
0.5
1.0
1.5
2.0
2.5
CyclinD2 P
Fold
enrichm
ent/Ig
G/C
ontr
ol r
egio
n BRG1
BRM
ns
ns✱✱✱
✱✱✱
.CC-BY-NC 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint
Graphical Summary:
Fig. 8 Graphical summary showing the effects of PFI-3 induced bromodomain inhibition on skeletal myogenesis. In normal
conditions, BRG1/BRM with active bromodomains can bind to promoters of target genes when muscle differentiation is
induced. This in turn affects two important aspects of skeletal myogenesis: cell cycle exit and the formation of
differentiated multinucleated myotubes. In the presence of PFI-3, BRG1 and BRM show reduced binding to target gene
promoters leading to continued cell-cycle and incomplete differentiation resulting in shorter myotubes.
.CC-BY-NC 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 26, 2020. ; https://doi.org/10.1101/2020.08.25.267666doi: bioRxiv preprint